Irith Baumann

Impaired uptake of apoptotic cells into tingible body macrophages in germinal centers of patients with systemic lupus erythematosus

OBJECTIVEnTo investigate the fate of apoptotic cells in the germinal centers (GCs) of patients with systemic lupus erythematosus (SLE).nnnMETHODSnLymph node biopsy specimens obtained from 7 SLE patients with benign follicular hyperplasia, 5 non-SLE patients with benign follicular hyperplasia (non-SLE), 5 patients with malignant follicular lymphoma, and 3 patients with dermatopathic lymphadenitis were stained with monoclonal antibodies against macrophages (CD68) and follicular dendritic cells (CR2/CD21). TUNEL staining and transmission electron microscopy were performed to detect apoptotic cells. Confocal microscopy was used to evaluate the in vivo capacity of tingible body macrophages to remove apoptotic cell material.nnnRESULTSnIn a subgroup of patients with SLE, apoptotic cells accumulated in the GCs of the lymph nodes. The number of tingible body macrophages, which usually contained engulfed apoptotic nuclei, was significantly reduced in these patients. In contrast to what was observed in all controls, TUNEL-positive apoptotic material from SLE patients was observed to be directly associated with the surfaces of follicular dendritic cells (FDCs).nnnCONCLUSIONnOur findings suggest that in a sub-group of SLE patients, apoptotic cells are not properly cleared by tingible body macrophages of the GCs. Consequently, nuclear autoantigens bind to FDCs and may thus provide survival signals for autoreactive B cells. This action may override an important control mechanism for B cell development, resulting in the loss of tolerance for nuclear antigens.

Exposure of anionic phospholipids serves as anti-inflammatory and immunosuppressive signal--implications for antiphospholipid syndrome and systemic lupus erythematosus.

In contrast to necrotic cells, the clearance of apoptotic ones usually is an anti-inflammatory process which elicits only a marginal immune response. During apoptosis phosphatidylserine (PS) is exposed on the outer leaflet of the cytoplasmic membrane and serves as target for the PS receptor of phagocytes. The latter is responsible for anti-inflammatory signalling and the induction of TGFbeta. We were interested whether the immunogenicity of apoptotic cells can be increased by masking PS. We observed that treatment of xenogeneic apoptotic cells with annexin V (AxV) significantly increased the humoral immune response against surface epitopes of these cells. Furthermore, AxV-coated irradiated tumour cells were able to elicit a long lasting tumour specific cytotoxic T lymphocyte response. AxV efficiently blocked the uptake of irradiated cells by macrophages but not by dendritic cells. Furthermore, AxV skewed the phagocytosis of irradiated cells towards inflammation. Investigation of patients with autoimmune diseases further supported the role of anionic surface phospholipids for anti-inflammatory clearance of apoptotic cells. Impaired clearance and opsonisation with anti-phospholipid-antibodies are discussed to be responsible for the development of systemic lupus erythematosus and anti-phospholipid-syndrome, respectively. Presentation of cryptic epitopes from late apoptotic cells in a proinflammatory context may challenge T cell tolerance. In addition, accumulation of uncleared apoptotic debris in the germinal centres of lymph nodes may result in the survival of autoreactive B cells.

Evaluation of dysplastic features in myelodysplastic syndromes: experience from the morphology group of the European Working Group of MDS in Childhood (EWOG-MDS)

In the absence of genetic abnormalities, the diagnoses of myelodysplastic syndromes (MDS) is primarily based on the presence of dysplasia in blood and marrow cells. Currently, there is no standardized approach to evaluate dysplasia. International cooperative study groups like the European Working Group on MDS in Childhood (EWOG-MDS) depend, however, on a concordance in diagnoses by their national reference centres for morphology. In EWOG-MDS, the morphological diagnoses of all cases enrolled from Scandinavia, the Netherlands, Germany, the Czech Republic, Austria and Italy are established by five experienced pathologists or hematologists cooperating in a morphology board. To study their concordance in evaluating myelodysplastic disorders, members of the morphology board initiated blinded reviews of smears of blood and bone marrow aspirates of known cases. Four features of dysplasia in granulopoiesis, erythropoiesis and megakaryopoiesis were assessed on May–Grünwald–Giemsa stained smears. In a final review of six blinded cases, good concordance for these features was achieved among the five observers. Accurately defined and restrictively applied cellular features of dysplasia are an important tool to improve and ensure the concordance in the diagnosis of MDS among investigators. For cooperative groups, agreement on the evaluation of the morphological assessment of dysplasia is a prerequisite.

Transcription of Cytokeratins 8, 18, and 19 in Bone Marrow and Limited Expression of Cytokeratins 7 and 20 by Carcinoma Cells: Inherent Limitations for RT-PCR in the Detection of Isolated Tumor Cells

The suitability of “real-time” quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of isolated carcinoma cells in bone marrow was investigated by evaluating the expression of cytokeratin (CK)7, CK8, CK18, CK19, and CK20 in 17 gastrointestinal cancer cell lines, 64 control bone marrow specimens from noncancer patients, and 30 bone marrow specimens from patients with gastric or colorectal cancer. RT-PCR products for CK8 and CK18 were detected in all cancer cell lines, but only 16, 5, and 11 cell lines provided evidence for CK19, CK7, and CK20 transcription. Variable numbers of bone marrow specimens from noncancer patients demonstrated background transcription of CK8 (78.1%), CK18 (95.3%), CK19 (35.9%), CK20 (29.6%), and CK7 (16.7%). Maximal background transcription for CK8, CK18, and CK19 ranged from 52.2 to 56.1 copies/103 copies glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the corresponding values of 0.06 and 0.76 copies for CK7 and CK20 being distinctly lower. When maximal background values were used as a threshold value to define positivity in tumor cell dilution experiments, sensitivity levels of one tumor cell in 104 bone marrow cells were determined for CK7 and CK20 RT-PCR assays. Maximal background expression values of the different CKs as obtained in the control series were exceeded once (CK20), twice (CK18 and CK19), and 18 times (CK7) in bone marrow specimens from cancer patients, with none of these specimens exceeding the maximal background expression value of CK8. We conclude that RT-PCR for CK8, CK18, and CK19 cannot be recommended for the detection of isolated tumor cells in bone marrow of cancer patients. On the other side, the limited number of gastric and colorectal cancer cell lines expressing CK7 and CK20 indicates that assay sensitivity for these CKs might be limited because of their selective expression by carcinoma cells.

Hodgkin's disease and peripheral T-cell lymphoma: composite lymphoma with evidence of Epstein-Barr virus infection.

This paper reports the case of a patient with a composite lymphoma consisting of nodular sclerosing Hodgkins disease and peripheral T‐cell lymphoma. The Hodgkin and Reed–Sternberg (HRS) cells harboured the Epstein–Barr virus (EBV) and displayed a type II EBV latency (LMP1+/EBNA2−), whereas the neoplastic T‐cells were EBV‐negative. Four years later, the patient presented with a relapse of the peripheral T‐cell lymphoma. In situ hybridization revealed numerous EBV‐carrying lymphocytes, which were shown to be polyclonal B‐cells with a latency III pattern of EBV gene expression (LMP1+/EBNA2+). This observation suggests that impairment of EBV‐specific immunity in the micro‐environment of T‐cell lymphomas may facilitate the outgrowth of EBV‐carrying B‐lymphocytes and emphasizes the importance of determining the phenotype of EBV‐infected cells, particularly when studying T‐cell lymphomas. The results further suggest that the HRS cells and neoplastic T‐cells were of different clonal origins. The detection of EBV‐carrying cell populations admixed with the neoplastic T‐cells at primary presentation and at relapse raises the possibility that the growth of the T‐cell lymphoma was dependent on the presence of such cells. Copyright

Myelodysplastic features in an infant with cystic fibrosis presenting with anaemia, oedema and failure to thrive

Myelodysplastic changes in peripheral blood or bone marrow cells are observed in patients with a variety of heterogeneous non-malignant and malignant conditions such as viral infections, vitamin deficiencies, metabolic diseases, congenital bone marrow failure syndromes, myelodysplastic syndromes (MDS) and acute leukaemia. However, myelodysplastic features in the bone marrow of patients with cystic fibrosis (CF) have not yet been described. We describe an infant with CF presenting with anaemia. Bone marrow investigation revealed myelodysplastic changes in precursor cells of all three lineages. A 2.5-month-old breast-fed male born at term to non-consanguineous healthy white parents was referred to our hospital for assessment of poor weight gain, anaemia, oedema and hypoproteinaemia. At birth, he weighed 2850 g (10th–50th percentile) with a birth length of 50 cm (50th percentile). At the age of 8 weeks, the parents noted swelling of both upper eyelids and lower extremities. He was admitted to a local hospital where he was found to have a haemoglobin (Hb) concentration of 6.7 g/dl with a mean cellular volume (MCV) of 92 fl. The antiglobulin test was negative. Serum protein and albumin were 3.3 g/dl and 1.9 g/dl, respectively. Vitamin B6 and B12 levels were normal. He was treated with intravenous albumin and transferred to our hospital. Physical examination revealed a pale infant in no distress with oedema of both feet. He weighed 3640 g (360 g below the 3rd percentile) with a length of 54 cm (10th percentile). A chest X-ray film and abdominal ultrasound imaging were normal. Laboratory investigations revealedthe following: Hb 5.9 g/dl, MCV 95 fl, reticulocyte count 298·10/l, white blood count 12·10/l with a normal differential count (neutrophils 44%) and platelets 408·10/l. Red cell morphology showed anisoand poikilocytosis and polychromasia. The total bilirubin level was 2.0 mg/dl with a direct bilirubin level of 0.3 mg/dl. The lactate dehydrogenase concentration was normal. The bone marrow aspirate showed a reduced cellularity, moderate myelodysplastic changes of all three cell lines (Fig. 1) and a slight increase in blast percentage (8%). Although the described changes fulfilled the recently published minimal criteria for childhood MDS [2], failure to thrive with hypoproteinaemia suggested CF instead. The sweat test with an increased chloride level was diagnostic and mutation analysis uncovered a homozygous delta F508 mutation in the CFTR gene. Pancreatic enzyme replacement and supplementation

Morphologische hämatologische Diagnostik

Das Mikroskop war das wichtigste Instrument des Hamatologen bis in die Mitte des letzten Jahrhunderts. In den 60er-Jahren wurden Methoden der Zytogenetik und der Zellkultivierung eingefuhrt, das Philadelphia-Chromosom beschrieben und die CML als Stammzellerkrankung erkannt. Die beiden folgenden Jahrzehnte waren neuen molekulargenetischen Methoden, den Blots und der PCR, gewidmet. Heute reden wir von Oligos und Chips, am Himmel ziehen Proteomics und Degradomics auf. Ist das Mikroskop ein Ladenhuter? Wer wurde heute jungen Mitarbeitern empfehlen, sich schwerpunktmasig mit der Morphologie von Blutund Knochenmarkzellen zu beschaftigen? Und doch ist die Morphologie das Herzstuck der klinischen Hamatologie geblieben. Heute wie vor 50 Jahren will sie erarbeitet und ein Stuck erobert werden.